Carotenoids are 40-carbon (C.sub.40) terpenoids consisting generally of eight isoprene (C.sub.5) units joined together. Linking of the units is reversed at the center of the molecule. Trivial names and abbreviations will be used throughout this disclosure, with IUPAC-recommended semisystematic names given in parentheses after first mention of each name.
Carotenoids are pigments with a variety of applications. For example, beta-carotene (.beta.,.beta.-carotene) is widely known and used as provitamin A and margarine colorant.
Phytoene (7,8,11,12,7',8',11',12'-octahydro-.psi.,.psi.-carotene) is the first carotenoid in the carotenoid biosynthesis pathway and is produced by the dimerization of a 20-carbon atom precursor, geranylgeranyl pyrophosphate (GGPP). Phytoene has useful applications in treating skin disorders (U.S. Pat. No. 4,642,318) and is itself a precursor for colored carotenoids. Aside from certain mutant organisms, such as Phycomyces blakesleeanus carB, no current methods are available for producing phytoene via any biological process.
Current methods for commercial production of carotenoids include chemical synthesis, e.g. beta-carotene, and extraction from biomass, e.g. zeaxanthin.
Carotenoids are synthesized in a variety of bacteria, fungi, algae, and higher plants. At the present time only a few plants are widely used for commercial carotenoid production. However, the productivity of carotenoid synthesis in these plants is relatively low and the resulting carotenoids are expensively produced.
One way to increase the productive capacity of biosynthesis would be to apply recombinant DNA technology. Thus, it would be desireable to produce carotenoids generally and phytoene specifically by recombinant DNA technology. This would permit control over quality, quantity and selection of the most suitable and efficient producer organisms. The latter is especially important for commercial production economics and therefore availability to consumers. For example, yeast, such as S. cerevisiae in large fermentors and higher plants, such as alfalfa or tobacco, can be mobilized for carotenoid production as described hereinafter.
An organism capable of carotenoid synthesis and a potential source of genes for such an endeavor is Erwinia herbicola, which is believed to carry putative genes for carotenoid production on a plasmid (Thiry, J. Gen. Microbiol., 130:1623 (1984)) or chromosomally (Perry et al., J. Bacteriol, 168:607 (1986)). Erwinia herbicola is a genus of Gram-negative bacteria of the ENTEROBACTERIACEAE family, which are facultative anaerobes. Indeed, recently published European Patent Application 0 393 690 A1 (published Apr. 20, 1990; sometimes referred to herein as "EP 0 393 690") reports use of DNA from another Erwinia species, Erwinia uredovora 20D3 (ATCC 19321), for preparing carotenoid molecules.
As is discussed in detail hereinafter, the present invention utilizes DNA from Erwinia herbicola EHO-10 (ATCC 39368) for preparation of carotenoid molecules and the enzymes used in their synthesis. Erwinia herbicola EHO-10 used herein is also referred to as Escherichia vulneris.
The genus is commonly divided into three groups. Of the three, the Herbicola group includes species (e.g. Erwinia herbicola) which typically form yellow pigments that have now been found to be carotenoids.
These bacteria exist as saprotrophs on plant surfaces and as secondary organisms in lesions caused by many plant pathogens. They can also be found in soil, water and as opportunistic pathogens in animals, including man.
A precise organismic function has yet to be ascribed to the pigment(s) produced by Erwinia herbicola. Perry et al., J. Bacteriol., 168:607 (1986), showed that the genes coding for the production of an unknown yellow pigment lie within an approximately 13-kilobase (kb) sequence coding for at least seven polypeptides, and that the expression of the yellow pigment is cyclic AMP mediated. Tuveson, J. Bacteriol., 170:4675 (1988), demonstrated that these genes, cloned from Erwinia and expressed in an E. coli strain, offered the host some protection against inactivation by near-UV light and specific phototoxic molecules.
E. coli and S. cerevisiae are commonly used for expressing foreign genes, but to optimize yields and minimize technical maintenance procedures, it would be preferable to utilize a higher plant species.